A method for simultaneous detection of 11 drug and metabolite residues in aquatic products

Abstract

The embodiment of the present invention discloses a method for simultaneously detecting the residual amounts of 11 drugs and metabolites in aquatic products. The method comprises the following steps: using ammoniated ethyl acetate as an extraction solvent to obtain a sample solution, measuring the sample solution by liquid chromatography-tandem mass spectrometry, and then performing qualitative and quantitative analysis to obtain the residual amounts of 11 drugs and metabolites in aquatic products. The present invention extracts 11 drugs and metabolites simultaneously, saving the amount of reagents used and the amount of labor, shortening the detection cycle, and achieving simultaneous scanning of positive and negative ions of the 11 drugs in an ultra-high performance liquid chromatography-tandem mass spectrometer ESI source by optimizing instrument conditions and mobile phase ratios, thereby improving the efficiency of instrument use. The method also has the advantages of high sensitivity, wide linear range, high correlation coefficient, high recovery rate, and good precision.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of detection and analysis technology, and specifically to a method for simultaneously detecting the residues of 11 drugs and their metabolites in aquatic products. Background Art

[0002] Due to the limitation of detection method, the detection of amide alcohols, nitroimidazoles and diazepam drugs and metabolites thereof in aquatic products needs to use different detection standards such as GB / T 20756, GB / T 21318, SN / T 3235 and SN / T 1865, at least 4 pre-treatments need to be carried out, not only a large amount of reagents, time, manpower and financial funds are wasted, but also unfavorable for improving regulatory efficiency, and there is no current effective detection standard for tinidazole, the existing detection method matrix effect of diazepam and florfenicol amine is large, the problems such as poor repeatability. In order to solve these difficult problems, the present invention establishes a kind of high-throughput simultaneous determination of amide alcohols (chloramphenicol, thiamphenicol, florfenicol and its metabolite florfenicol amine), nitroimidazoles (ronidazole, tinidazole, metronidazole, dimenamidazole and its metabolite hydroxymetronidazole, hydroxymetronidazole) and diazepam in aquatic products, a total of 11 kinds of drugs and metabolites. Summary of the Invention

[0003] To this end, an embodiment of the present invention provides a method for simultaneously detecting the residues of 11 drugs and their metabolites in aquatic products.

[0004] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0005] The present invention provides a method for simultaneously detecting the residual amounts of 11 drugs and metabolites in aquatic products. The method comprises the following steps: using ammoniated ethyl acetate as an extraction solvent to obtain a sample solution, measuring the sample solution by liquid chromatography tandem mass spectrometry, and then performing qualitative and quantitative analysis to obtain the residual amounts of the 11 drugs and metabolites in the aquatic products; wherein the ammoniated ethyl acetate is a mixed solution of ammonia water and ethyl acetate; and the 11 drugs and metabolites are: chloramphenicol, thiamphenicol, florfenicol, florfenicol amide, metronidazole, tinidazole, dimenidazole, ronidazole, hydroxymetronidazole, hydroxymetronidazole, and diazepam.

[0006] The present invention has conducted extensive research on sample extraction solvents and found that ammoniated ethyl acetate can efficiently extract the target analytes, reduce analyte loss, reduce impurity content, and minimize the effects of interfering substances. Compared with other extraction solvents such as ethyl acetate and acetonitrile, it is more conducive to fully extracting chloramphenicol, thiamphenicol, florfenicol, and florfenicol amide from tissues, thereby ensuring the accuracy of the detection method. Compared with ammoniated acetonitrile, it can significantly reduce impurity content while ensuring maximum extraction of the above 11 drugs and metabolites, which is conducive to improving the sensitivity of the detection method.

[0007] Furthermore, the preparation of the sample solution specifically includes: adding a first ammonium ethyl acetate to the sample, vortexing, ultrasonicating, centrifuging, collecting the supernatant, adding a second ammonium ethyl acetate to the residue, vortexing, ultrasonicating, centrifuging, combining the supernatants, and then concentrating. The obtained concentrated solution is purified and fixed to obtain a sample solution; wherein the volume concentration of ammonia water in the first ammonium ethyl acetate or the second ammonium ethyl acetate is 0.1%-2%; and the weight-to-volume ratio of the sample to the first ammonium ethyl acetate and the second ammonium ethyl acetate is 5±0.05g:12.5-17.5mL:7.5-12.5mL.

[0008] The present invention further studied the extraction method of the sample solution and found that under the above-mentioned extraction conditions, the provided method is simple, fast and accurate when simultaneously detecting 11 drugs and metabolites, and is suitable for large-scale sample testing. At the same time, the detection requires fewer types of reagents and smaller amounts, which is conducive to reducing costs and is more environmentally friendly.

[0009] Furthermore, the vortexing is performed at 1500-3000 r / min for 5-10 min; the ultrasound is performed at a frequency of 30-50 KHz for 5-15 min; and the centrifugation is performed at 3000-6000 r / min for 5-10 min.

[0010] Furthermore, the purification specifically includes: dissolving the concentrate with a methanol aqueous solution with a volume fraction of 5-30%, refrigerated centrifuging at 3000-6000 r / min and 0-8° C. for 10-20 minutes, and absorbing the lower clear liquid and filtering it into a sampling vial with an aqueous needle filter.

[0011] Furthermore, liquid chromatography conditions: ACQUITY UPLC BEH C18 chromatographic column; mobile phase gradient elution conditions: 0.0-0.5 min, 5% mobile phase B; 0.5-3.0 min, 5%-95% mobile phase B, 3.0-4.0 min, 95% mobile phase B; 4.0-4.5 min, 95%-5% mobile phase B, 4.5-5.0 min, 5% mobile phase B; wherein, mobile phase A: 10 mmol / L ammonium formate aqueous solution, pH adjusted to 7.0-7.5, mobile phase B: methanol.

[0012] The present invention conducts a large number of studies on liquid chromatography conditions. The type of mobile phase significantly affects the HPLC peak shape and signal response. For example, when mobile phase A is water and mobile phase B is methanol, the peak shape of chloramphenicol, thiamphenicol, florfenicol, tinidazole, metronidazole, ronidazole, dimernidazole, hydroxymetronidazole, and diazepam is better, but the peak shape of florfenicol amine and hydroxymetronidazole is poor; when mobile phase A uses a volume fraction of 0.1% formic acid aqueous solution and mobile phase B is methanol, the peak shape of florfenicol amine and hydroxymetronidazole becomes better, but the signal response of chloramphenicol, thiamphenicol and florfenicol in negative ion scanning mode is suppressed. The study found that under the above-mentioned liquid chromatography conditions, by taking into account the peak shape and signal response of 11 drugs and metabolites at the same time.

[0013] Furthermore, the specifications of the chromatographic column are: 50mm×2.1mm, and the particle size is 1.7μm.

[0014] Furthermore, the liquid chromatography conditions also include: column temperature: 35-45°C; flow rate: 0.20-0.50 mL / min; injection volume: 5.0-10.0 μL

[0015] Furthermore, the mass spectrometry conditions were as follows: ionization mode: electrospray ionization source (ESI), multiple reaction monitoring positive / negative ion scan mode (MRM); desolvation temperature: 400°C; desolvation gas flow rate: 1000 L / h; collision gas flow rate: 0.18 mL / min, cone gas flow rate: 150 L / h; ion source temperature: 400°C; mass spectrometry acquisition parameters for 11 drugs and metabolites, as well as their internal standards, are as follows:

[0016]

[0017]

[0018] Q: quantitative ion, q: qualitative ion.

[0019] Furthermore, the internal standard method was used for quantitative analysis, and the residual amounts of 11 drugs and metabolites in the aquatic products were calculated according to the following formula (1):

[0020]

[0021] Where:

[0022] X——the amount of the substance remaining in the sample, in micrograms per kilogram (μg / kg);

[0023] C s - the concentration of the analyte in the matrix standard working solution, in nanograms per milliliter (ng / mL);

[0024] A——chromatographic peak area of the analyte in the sample solution;

[0025] A s ——Chromatographic peak area of the analyte in the matrix standard working solution;

[0026] C i ——The concentration of the internal standard in the sample solution, in nanograms per milliliter (ng / mL);

[0027] C si - the concentration of the internal standard in the matrix standard working solution, in nanograms per milliliter (ng / mL);

[0028] A si —Chromatographic peak area of the internal standard in the matrix standard working solution;

[0029] A i ——Chromatographic peak area of internal standard in sample solution;

[0030] V——the final volume of the sample solution, in milliliters (mL);

[0031] m is the mass of the sample represented by the sample solution, in grams (g).

[0032] The embodiments of the present invention have the following advantages:

[0033] 1. The present invention uses 11 isotope internal standards for drugs and metabolites for quantification, which solves the problem of matrix effect in the original detection method and makes quantification more accurate.

[0034] 2. The present invention extracts 11 drugs and metabolites simultaneously, saving reagent usage, labor, and shortening the detection cycle.

[0035] 3. By optimizing the instrument conditions and using a 10 mmol / L ammonium formate aqueous solution (pH adjusted to 7.0-5.5) and methanol gradient elution, the present invention achieved simultaneous scanning of positive and negative ions in the ESI source of an ultra-high performance liquid chromatography-tandem mass spectrometer, thereby improving the efficiency of the instrument.

[0036] 4. The detection method provided by the present invention has high sensitivity to 11 drugs and their metabolites, a wide linear range, a high correlation coefficient, a high recovery rate and good precision. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0038] Figure 1 The mass chromatogram of chloramphenicol and its internal standard characteristic ion provided by the present invention;

[0039] Figure 2 The mass chromatogram of thiamphenicol and its internal standard characteristic ion provided by the present invention;

[0040] Figure 3 The mass chromatogram of florfenicol and its internal standard characteristic ion provided by the present invention;

[0041] Figure 4 The florfenicol amide and its internal standard characteristic ion mass chromatogram provided by the present invention;

[0042] Figure 5 The mass chromatogram of diazepam and its internal standard characteristic ion provided by the present invention;

[0043] Figure 6 The mass chromatogram of dimetridazole and its internal standard characteristic ion provided by the present invention;

[0044] Figure 7 The mass chromatogram of hydroxy-metronidazole and its internal standard characteristic ion provided by the present invention;

[0045] Figure 8 The mass chromatogram of metronidazole and its internal standard characteristic ion provided by the present invention;

[0046] Figure 9 The mass chromatogram of hydroxymetronidazole and its internal standard characteristic ion provided by the present invention;

[0047] Figure 10 The mass chromatogram of ronidazole and its internal standard characteristic ion provided by the present invention;

[0048] Figure 11 The present invention provides a mass chromatogram of tinidazole and its internal standard characteristic ion. DETAILED DESCRIPTION

[0049] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0050] Example 1

[0051] 1. Reagents and Materials

[0052] Unless otherwise specified, all reagents used below are of analytical grade, and the water used is first-grade water in accordance with GB / T 6682.

[0053] 1.1 Reagents

[0054] 1.1.1 Methanol (CH3OH): chromatographically pure.

[0055] 1.1.2 Hexane (C6H 14 ): chromatographically pure.

[0056] 1.1.3 Ammonium formate (NH4CHO2): high purity.

[0057] 1.1.4 Ammonia water (NH3·H2O), concentration is 25% NH3.

[0058] 1.1.5 1% aqueous ammonia-ethyl acetate solution (V / V): Accurately transfer 10 mL of aqueous ammonia to a 1000 mL volumetric flask and dilute to the mark with ethyl acetate. Prepare the solution immediately before use.

[0059] 1.1.6 10mmol / L ammonium formate aqueous solution: Weigh 0.63g of ammonium formate, dissolve to 1000mL, adjust the pH to 7.0-7.5, and prepare before use.

[0060] 1.2 Standard substances

[0061] 1.2.1 Chloramphenicol, thiamphenicol, florfenicol, florfenicol amide, metronidazole, tinidazole, dimernidazole, ronidazole, hydroxymetronidazole, hydroxymetronidazole, and diazepam, purity ≥ 99%.

[0062] 1.2.2 Chloramphenicol-D5, thiamphenicol-D3, florfenicol-D3, florfenicol amide-D3, metronidazole-D3, tinidazole-D5, dimenamidazole-D3, ronidazole-D3, hydroxymetronidazole-D3, hydroxymetronidazole-D3, diazepam-D5, purity ≥95%.

[0063] 1.3 Preparation of standard solution

[0064] 1.3.1 Standard stock solution: Accurately weigh appropriate amounts of each standard substance (equivalent to 10 mg of each active ingredient) of chloramphenicol, thiamphenicol, florfenicol, florfenicol amide, metronidazole, tinidazole, dimenidazole, ronidazole, hydroxymetronidazole, hydroxymetronidazole, and diazepam. Dissolve each substance in appropriate amounts of methanol and make up to the volume in a 100-mL volumetric flask to prepare a standard stock solution with a concentration of 100 μg / mL. Store below -18°C with a shelf life of 12 months.

[0065] 1.3.2 Internal Standard Stock Solution: Accurately weigh an appropriate amount of each of the internal standards (equivalent to 1 mg of each active ingredient) of chloramphenicol-D5, thiamphenicol-D3, florfenicol-D3, florfenicol amide-D3, metronidazole-D3, tinidazole-D5, dimethylnidazole-D3, ronidazole-D3, hydroxymetronidazole-D3, hydroxymetronidazole-D3, and diazepam-D5. Dissolve each in methanol and dilute to a 10 mL volumetric flask to prepare a standard stock solution at a concentration of 100 μg / mL. Store below -18°C. The shelf life is 12 months.

[0066] 1.3.3 Mixed Standard Intermediate Solution: Accurately pipette 1.00 mL of the chloramphenicol standard stock solution and 5.00 mL of the standard stock solutions of thiamphenicol, florfenicol, florfenicol amide, metronidazole, tinidazole, dimenidazole, ronidazole, hydroxymetronidazole, hydroxymetronidazole, and diazepam into a 100 mL volumetric flask. Dissolve to volume with methanol to prepare a mixed standard intermediate solution with a chloramphenicol concentration of 1.00 μg / mL and a thiamphenicol concentration of 5.00 μg / mL for thiamphenicol, florfenicol, florfenicol amide, metronidazole, tinidazole, dimenidazole, ronidazole, hydroxymetronidazole, hydroxymetronidazole, and diazepam. Store below -18°C. Valid for 6 months.

[0067] 1.3.4 Mixed internal standard intermediate solution: Accurately pipette 1.00 mL of chloramphenicol-D5 stock solution and 5.00 mL of internal standard stock solutions of thiamphenicol-D3, florfenicol-D3, florfenicol amide-D3, metronidazole-D3, tinidazole-D5, dimenidazole-D3, ronidazole-D3, hydroxymetronidazole-D3, hydroxymetronidazole-D3, and diazepam-D5 into a 50 mL volumetric flask and dilute to the mark with methanol to prepare a mixed internal standard intermediate solution with a chloramphenicol concentration of 2.00 μg / mL and a thiamphenicol-D3, florfenicol-D3, florfenicol amide-D3, metronidazole-D3, tinidazole-D5, dimenidazole-D3, ronidazole-D3, hydroxymetronidazole-D3, hydroxymetronidazole-D3, and diazepam-D5 concentration of 10.0 μg / mL. Store below -18℃, valid for 6 months.

[0068] 1.3.5 Mixed Standard Working Solution: Accurately pipette 1.00 mL of the mixed standard intermediate solution into a 100 mL volumetric flask and dilute to the mark with 15% methanol / water solution to prepare a mixed standard working solution with a chloramphenicol concentration of 10.0 ng / mL and thiamphenicol, florfenicol, florfenicol amide, metronidazole, tinidazole, dimernidazole, ronidazole, hydroxymetronidazole, hydroxymetronidazole, and diazepam at a concentration of 50.0 ng / mL. Store at 4°C in the dark. The shelf life is 3 months.

[0069] 1.3.6 Mixed Internal Standard Working Solution: Accurately pipette 5.00 mL of the mixed internal standard intermediate solution into a 100 mL volumetric flask and dilute to the mark with 15% methanol in water. This results in a mixed standard working solution containing 50.0 ng / mL of chloramphenicol-D5 and 250 ng / mL of thiamphenicol-D3, florfenicol-D3, florfenicol amide-D3, metronidazole-D3, tinidazole-D5, dimetridazole-D3, ronidazole-D3, hydroxymetronidazole-D3, hydroxymetronidazole-D3, and diazepam-D5. Store at 4°C in the dark. The shelf life is 3 months.

[0070] 2. Instruments and Equipment

[0071] 2.1 Ultra-high performance liquid chromatography-tandem mass spectrometer: Waters XEVO TQ-XS, equipped with an electrospray ion source.

[0072] 2.2 Balance: sensitivity 0.01g and 0.0001g.

[0073] 2.3 High-speed refrigerated centrifuge: suitable for 50mL and 15mL rotors.

[0074] 2.4 Nitrogen blowdown device: can accurately control temperature.

[0075] 2.5 Vortex mixer.

[0076] 2.6 Ultrasonic cleaner.

[0077] 2.7 Homogenizer.

[0078] 3. Methods and Steps

[0079] 3.1 Sample preparation

[0080] Purchase haddock, carp and prawns from the market, and prepare samples according to the requirements of Appendix B of GB / T 30891-2014.

[0081] Take the homogenized sample as the test sample;

[0082] Take the blank sample after homogenization as the blank test sample;

[0083] Take the blank sample after homogenization and add the standard intermediate solution of appropriate concentration to serve as the blank spiked sample.

[0084] 3.2 Extraction

[0085] Take 5 g of the sample (accurately weighed to ± 0.05 g) in a 50 mL centrifuge tube, add 200 μL of the mixed internal standard working solution, add 15 mL of 1% ammoniated ethyl acetate (V / V), vortex mixer at 2500 r / min for 5 min, ultrasonicate at 40 kHz for 10 min, centrifuge at 4000 r / min for 5 minutes, and transfer the supernatant to a 25 mL colorimetric tube; add 7.5 mL of 1% ammoniated ethyl acetate (V / V) at 2500 r / min for 5 min to the residue, ultrasonicate at 40 kHz for 10 min, centrifuge at 4000 r / min for 5 minutes, combine the supernatants into a 25 mL colorimetric tube, make up to 25.00 mL, mix well, and set aside.

[0086] 3.3 Concentration

[0087] Accurately transfer 5.00 mL of the above extract and blow dry with nitrogen at 40°C.

[0088] 3.4 Purification and solubility determination

[0089] Accurately add 2.00 mL of 15% methanol aqueous solution (v / v) to dissolve the residue dried in 3.3. Centrifuge at 4000 r / min and 0°C for 15 min. Pipette the lower supernatant and filter it with an aqueous syringe filter into an injection vial for determination by liquid chromatography-tandem mass spectrometry.

[0090] 4. Determination

[0091] 4.1 Liquid chromatography conditions

[0092] An ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm, particle size 1.7 μm) was used; the mobile phase gradient elution conditions are detailed in Table 1; mobile phase A: 10 mmol / L ammonium formate aqueous solution (pH adjusted to 7.0–7.5); mobile phase B: methanol; column temperature: 40°C; flow rate: 0.30 mL / min; injection volume: 10.0 μL.

[0093] Table 1 Mobile phase gradient elution program

[0094]

[0095]

[0096] 4.2 Mass spectrometry conditions

[0097] Ionization mode: electrospray ionization (ESI), multiple reaction monitoring positive / negative scan mode (MRM); desolvation temperature: 400°C; desolvation flow rate: 1000 L / h; collision gas flow rate: 0.18 mL / min, cone gas flow rate: 150 L / h; ion source temperature: 400°C; mass spectrometry acquisition parameters for the 11 drugs are shown in Table 2.

[0098] Table 2 UPLC-MS / MS mass spectrometry parameters for 11 drugs, their metabolites, and their internal standards

[0099]

[0100]

[0101] Q: quantitative ion, q: qualitative ion.

[0102] 4.3 Liquid chromatography-tandem mass spectrometry

[0103] 4.3.1 Qualitative determination

[0104] For each component to be measured, one parent ion and two or more daughter ions are selected. Under the same experimental conditions, the retention time ratio of the analyte and the internal standard in the sample, that is, the relative retention time, should be within ±2.5% of the corresponding relative retention time in the standard solution. The relative abundance of the qualifier ions of each component in the sample should be compared with the relative abundance of the corresponding qualifier ions in the standard solution with similar concentrations. If the deviation does not exceed the range specified in Table 3, it can be determined that the corresponding analyte is present in the sample.

[0105] Table 3 Maximum allowable deviation of relative ion abundance in qualitative confirmation (%)

[0106] Relative ion abundance ＞50 ＞20～50 ＞10～20 ＜10 Maximum allowed deviation ±20 ±25 ±30 ±50

[0107] 4.3.2 Quantitative determination

[0108] Under the instrument's optimal operating conditions, inject the mixed standard working solution. Draw a standard working curve using the ratio of the peak area of the analyte in the standard solution to the peak area of the isotope internal standard as the ordinate, and the ratio of the concentration of the analyte in the standard solution to the concentration of the corresponding isotope internal standard as the abscissa. Quantify the sample using the standard working curve. The response values of the analyte in the sample solution should all be within the linear range of the instrument. Internal standard quantification.

[0109] 5 Result calculation

[0110] The results were calculated according to the standard curve or formula (1):

[0111]

[0112] Where:

[0113] X——the amount of the substance remaining in the sample, in micrograms per kilogram (μg / kg);

[0114] C s - the concentration of the analyte in the matrix standard working solution, in nanograms per milliliter (ng / mL);

[0115] A——chromatographic peak area of the analyte in the sample solution;

[0116] A s ——Chromatographic peak area of the analyte in the matrix standard working solution;

[0117] C i ——The concentration of the internal standard in the sample solution, in nanograms per milliliter (ng / mL);

[0118] C si - the concentration of the internal standard in the matrix standard working solution, in nanograms per milliliter (ng / mL);

[0119] A si —Chromatographic peak area of the internal standard in the matrix standard working solution;

[0120] A i ——Chromatographic peak area of internal standard in sample solution;

[0121] V——the final volume of the sample solution, in milliliters (mL);

[0122] m——The mass of the sample represented by the sample solution, in grams (g);

[0123] Note: The blank value should be deducted from the calculation results.

[0124] 6. Sensitivity, accuracy and precision of the method

[0125] 6.1 Sensitivity

[0126] The detection limit for chloramphenicol in aquatic products using this method is 0.1 μg / kg, and the limit of quantification is 0.2 μg / kg. The detection limit for thiamphenicol, florfenicol, florfenicol amide, metronidazole, tinidazole, dimernidazole, ronidazole, hydroxymetronidazole, hydroxymetronidazole, and diazepam is 0.5 μg / kg, and the limit of quantification is 1.0 μg / kg. The results demonstrate that the sensitivity of this method meets the national detection limit requirements for these 11 veterinary drugs and their metabolites.

[0127] 6.2 Linear range

[0128] Under the optimized conditions, a series of standard solutions were prepared and sample injection analysis showed that the 11 target analytes showed good linear relationships in both positive and negative ion modes. The linear range of chloramphenicol was 0.2 μg / kg to 4 μg / kg, and the linear range of thiamphenicol was 1.0 μg / kg to 100 μg / kg. The linear ranges of florfenicol and florfenicol amide were both 1.0 μg / kg to 2000 μg / kg. The linear ranges of metronidazole, tinidazole, dimenidazole, ronidazole, hydroxymetronidazole, hydroxymetronidazole, and diazepam were all 1.0 μg / kg to 40 μg / kg, and the correlation coefficients (r) were all not less than 0.996 (Table 4).

[0129] Table 4 Standard working curves and correlation coefficients of 11 veterinary drugs

[0130]

[0131] Y is the ratio of the peak area of the analyte to the peak area of the internal standard, and X is the concentration (μg·L -1 ).

[0132] The results show that this method has a wide linear range and a high correlation coefficient, which fully meets the detection requirements.

[0133] 6.3 Accuracy

[0134] In this method, the recovery rate of chloramphenicol at the addition level of 0.2μg / kg to 4μg / kg is 70% to 120%; the recovery rate of thiamphenicol at the addition level of 1.0μg / kg to 100μg / kg is 70% to 120%; the recovery rate of florfenicol and florfenicol amide at the addition level of 1.0μg / kg to 2000μg / kg is 70% to 120%; the recovery rate of metronidazole, tinidazole, dimenidazole, ronidazole, hydroxymetronidazole, hydroxymetronidazole and diazepam at the addition level of 1.0μg / kg to 40μg / kg is 70% to 120%.

[0135] The results showed that this method had high recovery rates for 11 drugs and their metabolites and could meet the detection requirements at common test sample concentrations.

[0136] 6.4 Precision

[0137] The intra-batch repeatability of this method is ≤15%, and the inter-batch repeatability is ≤20%.

[0138] The results showed that this method had good precision and could meet the detection requirements.

[0139] Example 2

[0140] Mixed standard solutions of different concentrations were prepared based on 11 drugs and their metabolites. Blank samples of haddock, carp, and shrimp were used as matrices, and 11 compounds were added to perform three different level addition recovery experiments at low, medium, and high levels. Three replicates were determined for each concentration of different compounds. The results are shown in Table 5. The addition amount of chloramphenicol was 0.3 μg kg -1 , 1.0 μg kg -1 , 4.0 μg kg -1 Three different concentration levels, the other 10 compounds were added at 1.5 μg kg -1 , 5.0 μg kg -1 , 20.0 μg kg -1 At three different concentration levels, the average recoveries of 11 compounds ranged from 88.6% to 113.0%, with relative standard deviations of 0.2% to 11.5%, indicating that both the recovery and precision of this method meet the requirements for veterinary drug residue detection.

[0141] Table 5 Recovery rates of 11 compounds spiked (n=3)

[0142]

[0143] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for simultaneously detecting the residues of 11 drugs and their metabolites in aquatic products, characterized in that: Ammoniated ethyl acetate was used as the extraction solvent to obtain a sample solution, which was then measured by liquid chromatography-tandem mass spectrometry. Qualitative and quantitative analysis was then performed to determine the residues of 11 drugs and their metabolites in aquatic products. Wherein, the ammoniated ethyl acetate is a mixed solution of ammonia water and ethyl acetate; The 11 drugs and their metabolites are: chloramphenicol, thiamphenicol, florfenicol, florfenicol amide, metronidazole, tinidazole, dimernidazole, ronidazole, hydroxymetronidazole, hydroxymetronidazole, and diazepam; Liquid chromatography conditions: ACQUITY UPLC BEH C18 column; Mobile phase gradient elution conditions: 0.0-0.5 min, 5% mobile phase B; 0.5-3.0 min, 5%-95% mobile phase B, 3.0-4.0 min, 95% mobile phase B; 4.0-4.5 min, 95%-5% mobile phase B, 4.5-5.0 min, 5% mobile phase B; mobile phase A: 10 mmol / L ammonium formate aqueous solution, pH adjusted to 7.0-7.5, mobile phase B: methanol; The specifications of the chromatographic column are: 50 mm×2.1 mm, and the particle size is 1.7 μm.

2. The method for simultaneously detecting 11 drug and metabolite residues in aquatic products according to claim 1, characterized in that: The preparation of the sample solution specifically includes: adding a first ethyl ammonium acetate to the sample, vortexing, ultrasonicating, centrifuging, collecting the supernatant, adding a second ethyl ammonium acetate to the residue, vortexing, ultrasonicating, centrifuging, combining the supernatants, concentrating, purifying and fixing the obtained concentrated solution to obtain the sample solution; wherein, The volume concentration of aqueous ammonia in the first ethyl aminate or the second ethyl aminate is 0.1%-2%; The weight volume ratio of the sample to the first ethyl aminated ethyl acetate and the second ethyl aminated ethyl acetate is 5±0.05 g:12.5-17.5 mL:7.5-12.5 mL.

3. The method for simultaneously detecting 11 drug and metabolite residues in aquatic products according to claim 2, characterized in that: The vortexing is performed at 1500-3000 r / min for 5-10 min; the ultrasound is performed at a frequency of 30-50 KHZ for 5-15 min; and the centrifugation is performed at 3000-6000 r / min for 5-10 min.

4. The method for simultaneously detecting 11 drug and metabolite residues in aquatic products according to claim 2, characterized in that: The purification specifically includes: The concentrate was dissolved in a methanol aqueous solution with a volume fraction of 5-30%, and the mixture was centrifuged at 3000-6000 r / min and 0-8° C. for 10-20 min. The lower clear liquid was aspirated and filtered through an aqueous needle filter into a sample vial.

5. The method for simultaneously detecting 11 drug and metabolite residues in aquatic products according to claim 4, characterized in that: The liquid chromatography conditions also include: Column temperature: 35-45°C; flow rate: 0.20-0.50 mL / min; injection volume: 5.0-10.0 μL.

6. The method for simultaneously detecting 11 drug and metabolite residues in aquatic products according to claim 1, characterized in that: Mass spectrometry conditions are: Ionization mode: electrospray ionization source, multiple reaction monitoring positive / negative ion scan mode; desolvation gas temperature: 400°C; desolvation gas flow rate: 1000 L / h; collision gas flow rate: 0.18 mL / min, cone gas flow rate: 150 L / h; ion source temperature: 400°C; mass spectrometry acquisition parameters for 11 drugs and metabolites, as well as their internal standards, are as follows: Q: quantitative ion, q: qualitative ion.

7. The method for simultaneously detecting 11 drug and metabolite residues in aquatic products according to claim 1, characterized in that: The internal standard method was used for quantitative analysis, and the residual amounts of 11 drugs and their metabolites in the aquatic products were calculated according to the following formula (1): Where: X——the residual amount of the substance being tested in the sample, in micrograms per kilogram; C s - the concentration of the analyte in the matrix standard working solution, in nanograms per milliliter; A——chromatographic peak area of the analyte in the sample solution; A s ——Chromatographic peak area of the analyte in the matrix standard working solution; C i ——The concentration of the internal standard in the sample solution, in nanograms per milliliter; C si - the concentration of the internal standard in the matrix standard working solution, in nanograms per milliliter; A si —Chromatographic peak area of the internal standard in the matrix standard working solution; A i ——Chromatographic peak area of internal standard in sample solution; V——the final volume of the sample solution, in milliliters; m – the mass of the sample represented by the sample solution, in grams.

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